The present invention relates to a protein and its gene involved in the differentiation of testicular cells and belongs to the field of bioscience, specifically, developmental biology.
In the developmental process, reproductive cells carry out spermatogenesis via a differentiation process that includes meiosis. This differentiation process is different from that of somatic cells and consists of three main steps. The first step is the proliferation of spermatogenous cells and differentiation into primary spermatocytes. The second is the meiosis of primary spermatocytes, and the third is the transformation into sperms.
Owing to the progress in Molecular Biology, recent years have seen the isolation of several genes specifically expressed in these stages. For example, Hox-1.4 (Propst, F. et al. (1988) Oncogene 2:227-33), ferT (Sarge, K. D. et al. (1994) Biol Reprod 50:1334-1343) of the HSP70 family, and TESK1 (Toshima, J. et al. (1995) J. Biol. Chem. 270:31331-31337) that is a serine-threonine kinase, have been reported as genes specific to primary spermatocytes. However, still very little is known about the biological and physical roles of their gene products.
Genes expressing specifically in the differentiation process of reproductive cells carry a fundamental and vital role that decides the fate of those cells, and thus, defects in these genes are considered to be a cause of diseases such as infertility. Therefore, genes expressing specifically in the differentiation process of reproductive cells are recently gaining wide attention as targets in the development of pharmaceutical drugs. Such drugs can be used for the prevention and treatment of diseases such as infertility caused by defects in reproductive cell differentiation.
The present invention provides a novel protein relating to the differentiation of testicular cells, and the encoding gene. It also provides a vector and transformant used for, for example, producing the protein, and a method of producing the protein. The present invention also provides an oligonucleotide used for the isolation and the detection of the gene of the invention.
The inventors were evaluating the expression of genes encoding unknown proteins that trigger cell death when, irrelevant to their original aim, they unexpectedly succeeded in isolating a novel gene specifically expressed in the testis. When the databases were searched for the isolated gene, it was found to be a novel gene that did not have a significant homologous gene. Structural analysis of the protein encoded by the gene showed that it had in part a structure similar to the metal-binding site of metallothionein, which is known to be a metal-binding factor. Expression analysis in tissues revealed that the gene is extremely specific to the testis, especially to primary spermatocytes. The expression was not seen in the testis of infertile mice. Analysis of the human and mouse chromosomal locations showed that the gene was located in the same site as the gene locus that is known to be defective in infertile mice. Results of these analyses suggest that the protein encoded by the isolated gene is involved in regulating the differentiation of the testis.
The present invention relates to a novel protein involved in the regulation of testicular differentiation having a metal-binding site, and the gene thereof, more specifically:
(1) a protein comprising the amino acid sequence of SEQ ID NO: 4 or 5;
(2) a protein which comprises an amino acid sequence in which one or more amino acids in the amino acid sequence of SEQ ID NO: 4 or 5 have been replaced, deleted, and/or added, and which is functionally equivalent to the protein of (1);
(3) a protein which is encoded by a DNA hybridizing to the DNA comprising the nucleotide sequence of SEQ ID NO: 1 or 3, and which is functionally equivalent to the protein of (1);
(4) a DNA encoding the protein of any one of (1) to (3);
(5) a vector comprising the DNA of (4);
(6) a transformant comprising the DNA of (4) in an expressible manner;
(7) a method of producing the protein of any one of (1) to (3) comprising the steps of culturing the transformant of (6), and collecting the expressed protein from said transformant or the culture supernatant thereof;
(8) an antibody binding to the protein of any one of (1) to (3); and,
(9) a DNA specifically hybridizing to a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 3, and comprising at least 15 nucleotides.
The present invention provides the protein Tesmin, which may regulate the differentiation of spermatogenous cells into primary spermatocytes, and the gene thereof.
The inventors isolated two types of Tesmin cDNA of mouse origin arising possibly from splicing differences in the transcriptional process. The nucleotide sequences of these cDNAS are shown in SEQ ID NOs: 1 and 2, and the amino acid sequence of the protein encoded by these cDNAs in SEQ ID NO: 4. The nucleotide sequence of human Tesmin cDNA also isolated by the inventors is shown in SEQ ID NO: 3, and the amino acid sequence of the protein encoded by the cDNA is shown in SEQ ID NO: 5.
As shown in SEQ ID NOs: 1 and 2, mouse-derived Tesmin cDNA has an ORF encoding a protein comprising 295 amino acids. On the other hand, human-derived Tesmin cDNA has an ORF encoding a protein comprising 299 amino acids, as shown in SEQ ID NO: 3. SDS-PAGE analysis of the in vitro translational product of mouse Tesmin using 35S-labeled methionine showed that mouse Tesmin protein had a molecular weight of 32.5 kDa (FIG. 3).
Among the tissues within the body, both mouse and human Tesmin genes were expressed only in the testis, as revealed by Northern blot analysis and RT-PCR (FIGS. 1 and 2). RT-PCR analysis showed that Tesmin gene is hardly expressed in the immature testis up to day 8 following birth, but the expression increases from day 12 when the sperm differentiation starts, and its high expression stabilizes from day 18. In the W/Wv mouse known as an infertile mouse that lacks the growth factor receptor xe2x80x9cc-kitxe2x80x9d gene, Tesmin gene expression was hardly seen even in the matured testis of day 52 following birth (FIG. 4). These facts suggest that the Tesmin protein is involved in the differentiation of the testis. The Tesmin protein and its gene can be applied, for example, in the treatment of infertility.
The Tesmin protein of the invention can be prepared by incorporating DNA encoding the protein (e.g., DNA comprising the nucleotide sequence of any one of SEQ ID NO: 1 to 3) into a suitable vector, introducing this into a suitable host cell, and purifying the protein from the transformant obtained. The protein of the present invention can also be prepared as a recombinant protein made using genetic engineering techniques by culturing cells transformed with DNA encoding the Tesmin protein, as mentioned later. The natural protein can be isolated from testicular tissues by methods well known to one skilled in the art, for example, the affinity chromatography later described, using an antibody that binds to the Tesmin protein.
A skilled artisan can prepare not only a natural Tesmin protein, but also a modified protein functionally equivalent to the natural protein by, for example, suitably performing amino acid substitution of the protein using known methods. Amino acid mutations of a protein can occur spontaneously, too. Therefore, the protein of the invention includes a mutant in which the amino acid sequence of the natural protein was mutated by, for example, replacing, deleting, or adding one or several amino acids, and which is functionally equivalent to the natural protein. Methods well known to a skilled artisan for modifying amino acids are, for example, PCR-mediated site-specific-mutation-induction system (GIBCO BRL, Gaithersburg, Md.), oligonucleotide-mediated site-specific-mutagenesis (Kramer, W. and Fritz, H J (1987) Methods in Enzymol. 154:350-367), the Kunkel method (Methods Enzymol. 85:2763-2766 (1988)), and so on. The number of amino acids mutated is normally within ten amino acids, preferably within six amino acids, and more preferably within three amino acids.
Herein, xe2x80x9cfunctionally equivalentxe2x80x9d means that the mutant protein has a biochemical and/or biological activity equivalent to the natural protein. As such activities, for example, the binding activity between the protein and metal, and the testicular cell differentiation-inducing activity can be given.
The metal-binding activity can be detected, for example, as follows. First, the recombinant Tesmin protein is EDTA-treated to remove heavy metals that may be bound to the Tesmin protein. Next, EDTA is removed by gel filtration, and then, the heavy metals (for example, Zn2+, Cd2+, Cu2+, etc.) to be examined are added and reacted with the;recombinant Tesmin protein. After reacting, the presence or absence of a metal bond is detected as CD spectra using a CD spectropolarimeter (J-500C by Jasco) (refer Presta A. et al., Eur. J. Biochem Jan 15; 227(1-2):226-240).
The testicular cell differentiation-inducing activity can be detected, for example, as follows. First, spermatogoniums, spermatogenous cells, and primary spermatocytes are isolated from mouse testis by centrifugation. Next, Tesmin gene is incorporated into an expression vector (e.g., pBK-CMV vector, Stratagene), and the gene incorporated is introduced to cells isolated by lipofectAMINE (GIBCO BRL). After culturing the cells from a few hours to a few days, the expression of a genetic marker that identifies the differentiation stage (e.g., MEG1, ssH2B, etc.) is verified by the RT-PCR method.
The hybridization technique (Sambrook, J et al., Molecular cloning 2nded. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to a skilled artisan as an alternative method for isolating a functionally equivalent protein. In other words, it is a general -procedure for a skilled artisan to isolate DNA having a high homology to the whole or part of the DNA encoding the mouse or human Tesmin protein (a DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 3) and to obtain a protein functionally equivalent to the mouse or human Tesmin protein from the isolated DNA. Therefore, the protein of the present invention also includes a protein encoded by DNA hybridizing to DNA encoding the mouse or human-derived Tesmin protein, which is functionally equivalent to these proteins. When isolating the hybridizing DNA from other organisms, there is no restriction as to the organisms used, although testicular tissues from, for example, rats, rabbits, and cattle are suitable for the isolation. DNA isolated by hybridization techniques usually has a high homology to DNA encoding the mouse- and human-derived Tesmin protein (DNA comprising the nucleotide sequence of any one of SEQ ID NOs: 1 to 3). xe2x80x9cHigh homologyxe2x80x9d means, a sequence identity at the amino acid level of at least 40% or more, preferably 60% or more, more preferably 80% or more, and even more preferably, 95% or more. The homology of a sequence can be calculated, for example, by the method described in Proc. Natl. Acad. Sci. USA (1983) 80:726-730.
An example of hybridization conditions (stringent) for isolating a DNA high in homology is as follows. Namely, after conducting a prehybridization at 68xc2x0 C. for 30 min or more using the xe2x80x9cRapid-hyb bufferxe2x80x9d (Amersham LIFE SCIENCE), a labeled probe is added, and hybridization is done by incubating at 68xc2x0 C. for 1 hr or more. After that, washing is done three times within 2xc3x97SSC/0.01% SDS for 20 min at room temperature, and next, three times within 1xc3x97SSC/0.1% SDS, at 37xc2x0 C. for 20 min, followed by, two times within 1xc3x97SSC/0.1% SDS, at 50xc2x0 C. for 20 min.
This invention also provides a DNA encoding the Tesmin protein. The DNA of the present invention includes genomic DNA, synthetic DNA, and such, as well as cDNA, as long as such DNA encodes the Tesmin protein of the invention. The DNA of the invention can be used, for example, for producing recombinant proteins. Namely, the recombinant proteins can be prepared by inserting the DNA of the invention (e.g., SEQ ID NOs: 1 and 2) into a suitable expression vector, introducing this into a suitable cell, culturing the resulting transformant, and purifying the protein expressed. Cells used for the production of recombinant proteins are, for example, mammalian cells such as COS cells, CHO cells, and NIH3T3 cells; insect cells such as Sf9 cells; yeast cells; and E.coli, but there is no restriction as to the cells used. The vector for expressing the recombinant protein within cells varies according to the host cell, and, for example, pcDNA3 (Invitrogen), and pEF-BOS (Nucleic Acids. Res. 1990, 18 (17), p5322) and such are given as vectors for mammalian cells, Bac-to-BAC baculovirus expression system (GIBCO BRL) and such for insect cells, Pichia Expression Kit (Invitrogen) and such for yeast cells, and pGEX-5X-1 (Pharmacia) and QIAexpress system (Qiagen) and such for E.coli. Vectors can be introduced into hosts for example, by the calcium phosphate method, DEAE dextran method, the method using cationic liposome DOTAP (Boehringer Mannheim), electroporation method, calcium chloride method, etc. Transformants can be cultured according to their properties using methods well known to skilled artisans. Recombinant proteins can be purified from transformants by methods well known to skilled artisans, for example, the methods described in reference xe2x80x9cThe Qiaexpressionist handbook, Qiagen, Hilden, Germany.xe2x80x9d
The present DNA can be used for gene therapy of diseases caused by mutations of the gene. The Tesmin gene especially may be the causative of the genetic disease of infertile mice, and therefore, is expected to be applied in the gene therapy of infertility. When using for gene therapy, the DNA of the invention is inserted into, for example, a viral vector such as an adenovirus vector (e.g. pAdexLcw) and a retrovirus vector. (e.g. pZIPneo), or a non-viral vector, and administered to a target site of the body. The method of administration may be ex vivo or in vivo.
The present invention also provides an antibody that binds to the protein of the invention. The antibody of the present invention includes polyclonal antibodies and monoclonal antibodies. These antibodies can be prepared by following methods well known to skilled artisans. Polyclonal antibodies can be made by, for example, obtaining the serum of small animals such as rabbits immunized with the protein (or a partial peptide) of the present invention, and purifying by, for example, ammonium sulfate precipitation, a protein A or protein G column, etc. Monoclonal antibodies can be made by immunizing small animals such as mice with the protein (or a partial peptide) of the present invention, excising the spleen from the animal, homogenizing the organ into cells, fusing the cells with mouse myeloma cells using a reagent such as polyethylene glycol, selecting clones that produce antibodies against the protein of the invention from the fused cells (hybridomas), transplanting the obtained hybridomas into the abdominal cavity of a mouse, and collecting ascites from the mouse. The obtained monoclonal antibodies can be purified by, for example, ammonium sulfate precipitation, a protein A or protein G column, etc. The antibody thus prepared can be applied for antibody therapy and such, other than for the purification and detection of the protein of the invention. When administrating the antibody to humans with the aim of antibody therapy, humanized antibodies are effective in decreasing immunogenicity. Antibodies can be humanized by, for example, cloning the antibody gene from monoclonal antibody producing cells and using the CDR graft method which transplants the antigen-recognition site of the gene into a known human antibody. Human antibodies can also be prepared like ordinary monoclonal antibodies by immunizing a mouse whose immune system has been replaced by a human immune system with the protein of the invention.
This invention also provides a DNA specifically hybridizing to DNA encoding the Tesmin protein and comprising at least 15 nucleotides. The term xe2x80x9cspecifically hybridizingxe2x80x9d as used herein indicates that cross-hybridization does not significantly occur with DNA encoding proteins other than the Tesmin protein, under the usual hybridization conditions, preferably under stringent hybridization conditions. Such DNA can be used as a probe for detecting or isolating DNA encoding the Tesmin protein, or as a primer for amplification. Tesmin gene is expressed only in the testis, and even in the testis, it is expressed for a limited period. Therefore, the DNA can be used as a testis differentiation marker (a test drug). Also, there is a possibility that the Tesmin gene is the causative gene of the genetic disease of infertile mice, and therefore, the DNA may be used for the testing of infertility.